Suppression of Endogenous Immunoglobulin Expression in Non-Human Transgenic Animals

Abstract

The invention provides anovel approach for the suppression of endogenous antibody expression in non-human transgenic animals genetically engineered to express one or several human or humanized immunoglobulin transloci. Endogenous immunoglobulin expression in transgenic non-human animals is suppressed by selective expression of a suicide gene like a toxin only in B-cells expressing endogenous immunoglobulin but not in B-cells expressing human(ized) immunoglobulins. This method allows the dominant expression of transloci coding for humanized or human antibodies in the blood, milk and eggs of transgenic animals.

1 . A method for selective suppression of endogenous immunoglobulin production in B-cells of a non-human transgenic animal carrying an exogenous immunoglobulin translocus, comprising selectively expressing at least one suicide gene in B-cells producing an endogenous immunoglobulin of said non-human transgenic animal, but not in B cells producing an exogenous immunoglobulin, whereby B cells producing the endogenous immunoglobulin are depleted, and production of the endogenous immunoglobulin is suppressed, without suppressing the production of said exogenous immunoglobulin. 
     
     
         2 . The method of  claim 1  wherein said exogenous immunoglobulin is a human(ized) immunoglobulin heavy and/or light chain sequence. 
     
     
         3 . The method of  claim 1  or  claim 2  wherein said suicide gene, introduced into the B-cells of said non-human transgenic animal, is under the control of a B-cell specific promoter and is flanked by recombination sequences. 
     
     
         4 . The method of  claim 3  wherein said human(ized) immunoglobulin chain translocus is introduced into the B-cells of said non-human transgenic animal as part of an expression construct, additionally encoding a recombinase recognizing said recombination sequences, wherein expression of said suicide gene is inactivated through expression of said recombinase in B-cells expressing said human(ized) immunoglobulin translocus. 
     
     
         5 . The method of  claim 1  or  claim 2  wherein said suicide gene is selected from the group consisting of bacterial, fungal, insecticidal and plant toxins. 
     
     
         6 . The method of  claim 1  or  claim 2  wherein said suicide gene is a diphtheria toxin chain A. 
     
     
         7 . The method of  claim 1  or  claim 2  wherein said suicide gene is a prodrug converting enzyme. 
     
     
         8 . The method of  claim 7  wherein the prodrug converting enzyme is of non-mammalian origin. 
     
     
         9 . The method of  claim 8  wherein said prodrug converting enzyme of non-mammalian origin is selected from the group consisting of viral thymidine kinase (TK), bacterial cytosine deaminase (CD), bacterial carboxypeptidase G2 (CPG2), purine nucleotide phosphorylase (PNP), thymidine phosphorylase (TP), nitroreductase (NR), D-amino acid oxidase (DAAO), xanthine-guanine phosphoribosyl transferase (XGPRT), penicillin-G amidase (PGA), β-lactamase, multiple drug activation enzyme (MDAE), β-galactosidase (β-Gal), horseradish peroxidase (HRP) and deoxyribonucleotide kinase (DRNK). 
     
     
         10 . The method of  claim 7  wherein the prodrug converting enzyme is of human origin. 
     
     
         11 . The method of  claim 10  wherein the prodrug converting enzyme of human origin is selected from the group consisting of deoxycytidine kinase (dCK), carboxyesterases (CEs), carboxypeptidase A (CPA), β-glucuronidase (-Glu), and cytochrome P450 (CYP). 
     
     
         12 . The method of  claim 4  wherein said recombinase is selected from the group consisting of a Cre, Cre-like, Flp, φC31, λ integrase, phage R4 recombinase, TP901-1 recombinase, a prokaryotic transposase, a eukaryotic transposase, a viral retrotransposase, a  Drosophila  copia-like retrotransposase and a non-viral retrotransposase. 
     
     
         13 . The method of  claim 12  wherein said transposase or retrotransposase is selected from the group consisting of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn9, Tn10, Tn30, Tn101, Tn501, Tn903, Tn1000, Tn1681, Tn2901,  Drosophila  mariner, sleeping beauty transposase,  Drosophila  P element, maize Ac, Ds, Mp, Spm, En, dotted, Mu, I, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1) and Minos. 
     
     
         14 . The method of  claim 1  or  claim 2  wherein said non-human transgenic animal substantially stops antibody diversification by gene rearrangement early in life. 
     
     
         15 . The method of  claim 14  wherein said non-human transgenic animal substantially stops antibody diversification within the first month of its life. 
     
     
         16 . The method of  claim 1  or  claim 2  wherein said non-human transgenic animal is selected from the group consisting of rodents, rabbits, birds, cows, pigs, sheep, goats and horses. 
     
     
         17 . The method of  claim 16  wherein said rodent is a mouse or a rat. 
     
     
         18 . A transgenic expression construct comprising (1) a transgene, (2) a human or humanized immunoglobulin heavy and/or light chain translocus, (3) a self-cleaving peptide, and (4) a recombinase. 
     
     
         19 . A transgenic expression construct comprising (1) a transgene, and (2) a suicide gene that is under the control of a B-cell specific promoter, and is flanked by recombination sites recognized by a recombinase. 
     
     
         20 . A transgenic expression construct comprising:
 a first transgene further comprising a human or humanized immunoglobulin heavy and/or light chain locus, a self-cleaving peptide and a recombinase, and,   a second transgene further comprising a suicide gene that is under the control of a B-cell specific promoter, and is flanked by recombination sites recognized by said recombinase.   
     
     
         21 . The transgenic expression construct of  claim 18 ,  19  or  20  wherein said recombinase is a site specific recombinase selected from the group consisting of a Cre, Cre-like, Flp, φC31, λ integrase, phage R4 and TP901-1 recombinase. 
     
     
         22 . The transgenic expression construct of  claim 18 ,  19  or  20  wherein said recombinase is either a prokaryotic or a eukaryotic transposase. 
     
     
         23 . The transgenic expression construct of  claim 22  wherein said recombinase is either a viral,  Drosophila  copia-like or non-viral retrotransposons. 
     
     
         24 . The transgenic expression construct of  claim 23  wherein said retrotransposons is selected from the group consisting of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn9, Tn10, Tn30, Tn101, Tn501, Tn903, Tn1000, Tn1681, Tn2901 , Drosophila  mariner, sleeping beauty transposase,  Drosophila  P element, maize Ac, Ds, Mp, Spm, En, dotted, Mu, I, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1) and Minos. 
     
     
         25 . The transgenic expression construct of  claims 19  or  20  wherein said recombination sites are selected from a group consisting of a lox P site, FRT site, a bacterial genomic recombination site and a phage recombination site. 
     
     
         26 . The transgenic expression construct of  claim 25  wherein said bacterial genomic recombination site is attB and said phage recombination site is an attP or a pseudo-attP or a pseudo-attB site. 
     
     
         27 . The transgenic expression construct of  claims 18  or  20  wherein said self-cleaving peptide is obtained from viral 2A/2B or 2A-like/2B sequences. 
     
     
         28 . The transgenic expression construct of  claim 27  wherein said virus is selected from the group consisting of the picornaviridae virus family, the equine rhinitis A (ERAV) virus family, the picornavirus-like insect virus family or from the type C rotavirus family. 
     
     
         29 . The transgenic expression construct of  claim 27  wherein said virus is selected from the group consisting of the foot and mouth disease virus (FMDV), the equine rhinitis A (ERAV) virus, or the Thosea asigna virus (TaV). 
     
     
         30 . The transgenic expression construct of  claims 19  or  20  wherein said suicide gene is specifically expressed in B-cells using a promoter/enhancer selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD24, CD40, CD72, Blimp-1, CD79b, mb-1, tyrosine kinase bLk, VpreB, immunoglobulin kappa light chain, immunoglobulin lambda-light chain and immunoglobulin J-chain or modifications thereof. 
     
     
         31 . The transgenic expression construct of  claim 30  wherein said B-cell specific promoter/enhancer is the kappa light chain gene promoter or modifications thereof. 
     
     
         32 . A non-human transgenic animal expressing the transgenic expression constructs of  claims 18  and  19  or  20 . 
     
     
         33 . The non-human transgenic animal of  claim 32  that generates antibody diversity substantially by gene conversion. 
     
     
         34 . The non-human transgenic animal of  claim 32  that is selected from the group consisting of rodents, rabbits, birds including chickens, turkeys, ducks and geese. 
     
     
         35 . The non-human transgenic animal of  claim 34  that is either a mouse or a rat.